Limited Geographic Distribution of Beet Pseudo-Yellows Virus in Costa Rican Cucurbits

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Journal of Plant Pathology (2008), 90 (2), 331-335 Edizioni ETS Pisa, 2008 331

SHORT COMMUNICATION LIMITED GEOGRAPHIC DISTRIBUTION OF BEET PSEUDO-YELLOWS VIRUS IN COSTA RICAN CUCURBITS

P. Ramirez1, E. Hernandez1, F. Mora1, R. Abraitis2 and R.W. Hammond3

1 University of Costa Rica, San Jose, Costa Rica 2 Institute of Biotechnology, Vilnius, Lithuania 3 USDA-ARS, Molecular Plant Pathology Laboratory, Beltsville, MD, USA

SUMMARY In early 2004, severe yellowing and chlorotic (and oc- casionally mosaic) symptoms were observed in field- Beet pseudo-yellows virus (BPYV) was previously grown cucurbits in Costa Rica (Fig. 1A). Symptoms re- found to be associated with severe yellowing and chloro- sembled those of the genus Crinivirus and large popula- sis in field-grown cucurbits in Costa Rica. To determine tions of the greenhouse whitefly, Trialeurodes vaporario- the prevalence and molecular variability of BPYV in Cos- rum (Westwood), were observed in the fields and on ta Rica, leaf samples were collected in 2004 and 2005 symptomatic plants (Fig. 1B). To identify the causal from symptomatic cucurbit plants growing in geographi- agent of the disease, we performed reverse transcrip- cally distinct cucurbit-growing regions, and weedy plants tion-polymerase chain reaction (RT-PCR) analyses using growing adjacent to production fields. The samples were virus-specific primers corresponding to known cucur- tested for the presence of BPYV using RT-PCR with bit-infecting criniviruses and a flexivirus (Hammond et virus-specific primers and nucleic acid hybridization al., 2005). Amplified DNA fragments were obtained on- probes specific for genes encoding the BPYV minor coat ly in samples taken from symptomatic plants and con- protein, heat shock protein, and polymerase protein. Sev- taining either Beet pseudo-yellows virus (BPYV, Tzane- eral isolates were also amplified using primers spanning takis et al., 2003) or Cucumber yellows virus (CuYV, a an insertion region in RNA1 to examine isolate variabili- strain of BPYV, Hartono et al., 2003) primer sets; nu- ty. Our results revealed that (1) BPYV in cucurbits is cur- cleotide sequence analyses of purified PCR products rently limited to an isolated region of the country, (2) ge- verified their identity as variants of BPYV, with 97% netic variation in the genes examined among the isolates and 99% sequence identity with reported CPm and is very low, and (3) BPYV is present in wild cucurbit and HSP sequences, respectively. The two samples, obtained other plant species growing adjacent to production fields from Cucurbita moschata Duch. (ayote or squash) and containing BPYV-positive cucurbits. Cucurbita pepo L.(escalopini or sunburst squash, zucchini), were taken from a region around Paraiso, Cartago, Key words: Beet pseudo-yellows virus, BPYV, Crinivirus, Costa Rica (Hammond et al., 2005). Costa Rica. The current studies were undertaken to determine the prevalence and genetic variability of BPYV isolates Members of the whitefly-transmitted genus Crinivirus in major cucurbit-growing regions in Costa Rica. In ad- within the family Closteroviridae are emerging threats to dition, because of the wide host range of BPYV, we ex- both vegetable and fruit production worldwide (Tzane- amined weed species adjacent to BPYV-positive pro- takis and Martin, 2004a,b; Tzanetakis et al., 2003, 2006; duction fields as possible reservoirs of the virus. Wintermantel, 2004a,b; Wisler et al., 1998). Criniviruses Leaf samples were collected from 188 cucurbit plants, contain bipartite, single-stranded, message-sense RNA including ayote or squash, escalopini or sunburst squash, genomes, where RNA1 encodes the replication-associat- and zucchini, and Cucurbita melo L. (melon) on several ed proteins (including the methyltransferase, helicase and farms throughout Costa Rica, from January – September RNA-dependent RNA polymerase (POL), and RNA2 en- 2004, and in the Cartago/Paraiso region in 2005. The codes several open reading frames, including duplicated, plants showed general yellowing and/or chlorosis. The but divergent, coat protein genes (CPh and CPm) and a leaf samples were dried using silica gel. Total RNA was virus-encoded heat shock protein (HSP70) homolog, a extracted from leaf tissue using TRI Reagent (Molecular distinguishing feature of members of the Closteroviridae Research Inc., Cincinnati, OH, USA). RT-PCR reactions (Karasev, 2000; Martelli et al., 2002). containing one primer set at a time were performed with the Titan One-Tube RT-PCR kit (Roche Diagnostics Corp., Chicago IL, USA) using primers shown in Table 1 Corresponding author: R. Hammond for amplifying the minor coat protein gene (CPm) Fax: +1.301.5045449 E-mail: [email protected] (BPCPmF and BPCPmR) as previously described (Ham- 020_JPP_130SC_331_colore 21-07-2008 11:56 Pagina 332

332 BPYV in Costa Rica Journal of Plant Pathology (2008), 90 (2), 331-335

mond et al., 2005). Primers specific for the HSP70 gene HSP, and POL, selected RT-PCR products obtained (CYHSPF and CYHSPR) of the cucumber yellows virus above were cloned into the TOPO TA cloning vector strain of BPYV were designed based on published se- (Invitrogen, Carlsbad, CA, USA), sequenced to verify quence data (Table 1). In addition, degenerate primers their identity, and were labeled for nucleic acid hy- designed to anneal to conserved phosphate 1 and 2 mo- bridization using the DIG-High Prime DNA Labeling tifs of the HSP70 of whitefly-transmitted criniviruses Kit (Roche Diagnostics Corp.). Five µl aliquots of the (HSP-P-1 and HSP-P-2; Tian et al., 1996) and degener- total nucleic acids obtained from tissue samples were ate primers that amplify a conserved region in the POL spotted onto nylon membranes and hybridization was gene (Crini Pol F and Crini Pol R; Martin et al., 2001) performed at 42˚C using the DIG Easy Hyb buffer, fol- were used in RT-PCR experiments. We also examined the lowed by washing and colorimetric detection using the genetic diversity of the isolates around the unique inser- DIG Wash and Block Buffer set (Roche Diagnostics tion site at the end of the methyltransferase coding region Corp.). In 2004, when a more representative number of of RNA1 (Tzanetakis and Martin, 2004b) by designing samples were tested, more samples were positive by hy- primers that span the region nt 3029 - nt 3735 (BPYVR- bridization than by RT-PCR; however, in the 2005 sam- NA1F and BPYVRNA1R; Table 1). All RT-PCR reac- ples, a positive sample by RT-PCR could be negative by tions were performed using an annealing temperature of hybridization (Table 2). We observed that the POL hy- 42˚C and 35 cycles. bridization probe was not reliable in detecting virus in Of the 155 symptomatic samples collected in 2004 the test samples that were positive by RT-PCR. Our hy- from the major cucurbit-growing regions of the country, bridization results may reflect the notably low titer 34 (21%) were positive using both the BPYV CPm and BPYV in its host (Wisler et al., 1998). CYHSP primers (Table 2). The CPm primers did not Direct sequencing of all purified PCR products ob- detect as many positive samples as the CYHSP primers tained using CPm, HSP, and POL primer sets was per- (Table 2). The degenerate primers (HSP-P-1/HSP-P-2 formed using gene-specific primers. Alignment of the and Crini Pol F/Crini Pol R) did not identify positive nucleotide sequences and deduced amino acids was samples other than those already detected using the made using CLUSTAL W (Thompson et al., 2004). Phy- BPYV-specific primers, and in most cases, the degener- logenetic analyses were conducted using MEGA version ate primers did not detect samples that were positive 3.1 (Kumar et al., 2004). The respective nucleotide and with the BPYV-specific primers (data not shown). deduced amino acid sequences of the CPm, HSP, and Therefore, subsequent RT-PCR analyses were per- RNA1 PCR products of the individual isolates were 99- formed using both the BPYV CPm and CYHSP primer 100% identical to one another, in agreement with what pairs. In 2005, cucurbit samples were collected only in was previously found for the CPm gene of geographical- the Cartago/Paraiso region, and the incidence of BPYV ly distinct isolates of the crinivirus Cucurbit yellow stunt- was determined to be 93% using the CYHSP primers ing disorder virus (Rubio et al., 2001). and 48% using the CPm primers (Table 2). Phylogenetic analyses of CPm nucleic acid sequences Because RT-PCR may fail to detect virus-infection revealed that the BPYV isolates from Costa Rica are due to mismatches in the primer binding sites, the CPm, more closely related to the North American strawberry

Fig. 1. A. Symptomatic leaves of an older Cucurbita spp. plant in fields located in La Flor, Paraiso. B. Extensive populations of greenhouse whitefly on the lower surface of cucurbit leaves of plants grown in the affected region. 020_JPP_130SC_331_colore 21-07-2008 11:56 Pagina 333

Journal of Plant Pathology (2008), 90 (2), 331-335 Ramirez et al. 333

Table 1. Oligonucleotide primers used for PCR amplification and DNA sequencing.

Name Position* Primer sequence References BPCPmF 5241-5261 5’-TTCATATTAAGGATGCGCAGA-3’ Tzanetakis et al., 2003 BPCPmR 5553-5574 5’-TGAAAGATGTCCACTAATGATA-3’ Tzanetakis et al., 2003 CYHSPF 150-168 5’- GAGCGCCGCACAAGTCATC-3’ Hammond et al., 2005 CYHSPR 578-601 5’-TACCGCCACCAAAGTCATACATTA-3’ Hammond et al., 2005 BPYVRNA1F 2983-3004 5’-TTGAATGAATCGAAAGAGAACC-3’ this work, Tzanetakis and Martin, 2004b BPYVRNA1R 3778-3800 5’-GTGTTTTGAATCAAAGTCTCGAG-3’ this work, Tzanetakis and Martin, 2004b HSP-P-1 712-732 5’-GGNTTAGANTTCGGNACNAC-3’ Tian et al., 1996 HSP-P-2 1285-1305 5’TCAAANGTNCCNCCNCCNAA-3’ Tian et al., 1996 Crini Pol F 6377-6393 5’-GCYCCSAGRGTKAATGA-3’ Martin et al., 2001 Crini Pol R 6875-6892 5’-ACCTTGRGAYTTRTCAAA-3’ Martin et al., 2001

* Positions of 5’ and 3’ ends of primers with respect to the complete sequence of the BPYV RNA1 or RNA2 (GenBank accession numbers AY330918 and AY330919, respectively) or CYVHSP70 gene (primers CYHSPF and CYHSPR) (GenBank accession number AB085612).

Table 2. Comparison of BPYV detection methods.

Year collected1 HSP RT-PCR2 HSP HYB3 CP RT-PCR4 CP HYB5 2004 34 39 24 30 [155] (21%) (25%) (15%) (19%) 2005 31 20 16 9 [33] (93%) (60%) (48%) (27%)

1 In 2004, plants were collected from all major cucurbit-growing regions. In 2005, samples were collected from the Cartago/Paraiso region. Numbers in brackets represent the number of samples collected. 2 RT-PCR reactions were performed using the primers CYHSPF and CYHSPR at 42˚C. Numbers represent the number of BPYV-positive samples; numbers in parentheses are % of total samples collected that were positive. 3 Nucleic acid hybridization at 42˚C using a probe prepared from an RT-PCR product amplified using CYHSPF and CYHSPR primers 4 RT-PCR reactions were performed using the primers BPCPmF and BPCPmR at 42˚C. 5 Nucleic acid hybridization at 42˚C using a probe prepared from an RT-PCR product amplified using primers BPCPmF and BPCPmR.

isolate [AY330919; Tzanetakis and Martin, 2004b] than tion region, supporting previous evidence that these iso- to the cucurbit isolate from Japan [AB085612; Hartano lates are more related to the strawberry isolate. Phyloge- et al., 2003] (99.3% versus 97.5%, respectively) (Fig. netic analysis of the RNA1 sequences revealed that the 2). Most of the isolates appear to cluster into Group I, isolates do tend to cluster in geographically adjacent re- and represent samples taken from multiple sites adja- gions (data not shown). For example, the Cartago, Cer- cent and including the Cartago/Paraiso region. Similar vantes, and Paraiso isolates group together and not as results were found with the HSP gene (data not shown). separate clades. This suggests that replication-related Group II, whose members comprise samples also taken genes may be more conserved than CPm and HSP70 from the Cartago/Paraiso region (Fig. 2, designated by genes, and that a different selection pressure may be asterisks, respectively) contain two nucleotide changes placed on them. However, due to the low number of par- in the CPm sequence at nt 5139 and nt 5177. One of the simonius informative sites in the analyzed sequences (1% changes results in an amino acid change from D (Group (3/281nt) for CPm; 0.9% (7/752nt) for the RNA1 inser- I) to N (Group II) and the second change is silent. tion; 0.3% (1/327nt) for HSP70), it was difficult to re- Sequence analysis of a region in RNA1 found to con- solve the phylogenetic relationships among these isolates. tain an insertion of 49 amino acids in the strawberry iso- The current incidence of BPYV in cucurbits is re- late of BPYV (Tzantetakis and Martin, 2004b) and not stricted to a small area in the Central part of the country present in the cucurbit isolate of BPYV (Hartono et al., (in and near the Province of Cartago) (Fig. 3). This re- 2003) revealed that Costa Rican isolates contain the inser- gion is agriculturally rich and is located in the Central 020_JPP_130SC_331_colore 21-07-2008 11:56 Pagina 334

334 BPYV in Costa Rica Journal of Plant Pathology (2008), 90 (2), 331-335

Plateau. The source of the virus is unknown. Farmers near the production fields, or there may be infected plant cucurbit seeds, not seedlings, in production fields. wild cucurbit or other weed species that could serve as BPYV has a wide host range among crop and weed a reservoir of the virus. species (Duffus, 1965). Therefore, the source of inocu- To determine if virus reservoirs were present in or near lum may be symptomless ornamentals or perennials the infected fields, twenty-seven samples of weedy plants were collected in 2006 from areas adjacent to BPYV-infected fields in Cartago. In this preliminary survey, the plants were identified only to family and not to genus and species. Only the Cucurbitaceae sample was routinely positive for BPYV by RT-PCR. Sequence analysis of the RT-PCR product from the wild cucurbit sample, obtained using the BPYV CP primers, revealed that it is in Group I (sample number 3027) (Fig. 2). Samples from other plant families (Asteraceae, Balsamiaceae, Brassi- caceae, Caryophyllaceae, Portulacaceae, and Solanaceae) were also positive in one or more RT-PCR reactions, but not consistently or only weakly. This may be due to the lower titer of the virus in these plants. Sequence analysis of their RT-PCR products revealed their close relation- ship with the wild cucurbit isolate. The presence of BPYV in the wild species located in the affected area may provide a source of virus for infection of introduced crops, or, alternatively, the wild species may have ac- quired it from the field crop by insect transmission. Fur- ther investigation will provide more definitive informa- tion on the prevalence of BPYV in weedy species. Although plants exhibiting symptoms of yellowing and chlorosis were collected at all sampling sites, BPYV infection was restricted to the Cartago/Paraiso region. It is possible that these symptoms could be due to abi-

Fig. 2. Phylogenetic tree illustrating the relationships among the CPm sequences of the BPYV isolates. Forty taxa were compared for the CPm sequence of 281 nt. The analysis in- cluded truncated versions of the respective region of the strawberry isolate of BPVY [labeled Strawberry; AY330919; Tzanetakis and Martin, 2004b] or CuYV [labeled Cucumber; AB085612; Hartono et al., 2003], respectively. The taxa numbers on the tree represent the designation given the isolate when collected from the field. Samples #10-2554 were collected in 2004 and samples #2895-2929 were collected in 2005. Sample #3027 is the wild cucurbit sample. The tree was obtained using the Neighbor-Joining method and the pro- gram MEGA 3.1 as described in the text. The length of the Fig. 3. Map of Costa Rica showing major cities, and the loca- branches represents the genetic distance and the numbers at tion of collection sites that were negative () or positive () the branches represent the percentage of times in which this for the presence of BPYV by RT-PCR analysis. The arrow topology of the branch was observed after 5000 bootstrap designates the location of the Cartago region where all of the replicates. positive samples were collected. 020_JPP_130SC_331_colore 21-07-2008 11:56 Pagina 335

Journal of Plant Pathology (2008), 90 (2), 331-335 Ramirez et al. 335

otic factors, such as nutritional disorders, or to infection Martelli G.P., Agranovsky A.A., Bar-Joseph M., Boscia D., by other viruses (Wisler, 1998), although our attempts Candresse T., Coutts R.H.A., Dolja V.V., Falk B.W., Gon- to identify cucurbit-infecting potyviruses using universal salves D., Jelkmann W., Karasev A.V., Minafra A., Namba primers or Cucumber mosaic virus using virus-specific S., Vetten H.J., Wisler G.C., Yoshikawa N., 2002. The primers yielded negative results (data not shown). A family Closteroviridae revised. Archives of Virology 147: 2039-2044. further investigation into the cause of the BPYV-inde- Martin R.R., Tzanetakis I.E., Gergerich R., Fernandez G., pendent disease will be made in the future. Pesic S., 2001. Blackberry yellow vein associated virus: a BPYV was the first whitefly-transmitted closterovirus new crinivirus found in blackberry. Acta Horticulturae 656: identified and was discovered by J. E. Duffus in Califor- 137-142. nia greenhouses in 1965 (Duffus, 1965). Since that time, Rubio L., Abou-Jawdah Y., Lin H-X., Falk B.W., 2001. Geo- BPYV has been found in numerous locations world- graphically distant isolates of the crinivirus Cucurbit yellow wide, affecting many plant species. Although the eco- stunting disorder virus show very low genetic diversity in the nomic impact of BPYV on cucurbit production in Cos- coat protein gene. Journal of General Virology 82: 929-933. ta Rica has not yet been determined, it poses a new con- Thompson J.D., Higgins D.G., Gibson T.J., 2004. CLUSTAL cern for growers of both vegetable and ornamental W: improving the sensitivity of progressive multiple se- crops due to its wide host range. Management of the quence alignment through sequence weighting, positions- disease could include removal of weedy sources of virus specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673-4680. near production fields to minimize vector transmission. Tian T., Klaasen V.A., Soong J., Wisler G., Duffus J.E., Falk B.W., 1996. Generation of cDNAs specific to lettuce infec- tious yellows closterovirus and other whitefly-transmitted ACKNOWLEDGEMENTS viruses by RT-PCR and degenerate oligonucleotide primers corresponding to the closterovirus gene encoding the heat We want to acknowledge the excellent technical as- shock protein70 homolog. Phytopathology 86: 1167-1173. sistance of Sharon Jhingory and critical reading of the Tzanetakis I.E., Martin R.R., 2004a. First report of Beet pseu- manuscript by John Hammond. We also acknowledge I. do yellows virus in blackberry in the United States. Plant E. Tzanetakis for suggesting evaluation of sequence di- Disease 88: 223. versity in the unique insertion site in RNA1 of BPYV. Tzanetakis I.E., Martin R.R., 2004b. Complete nucleotide sequence of a strawberry isolate of Beet pseudoyellows virus. Virus Genes 28: 239-246. Tzanetakis I.E., Susaimuthu J., Gergerich R.C., Martin R.R., REFERENCES 2006. Nucleotide sequence of Blackberry yellow vein associated virus, a novel member of the Closteroviridae. Virus Duffus J.E., 1965. Beet pseudo-yellows virus, transmitted by Research 116: 196-200. the greenhouse whitefly (Trialeurodes vaporariorum). Phy- Tzanetakis I.E., Wintermantel W.M., Martin R.R., 2003. First topathology 55: 450-453. report of Beet pseudo yellow virus in strawberry in the Hammond R.W., Hernandez E., Mora F., Ramirez P., 2005. United States: A second crinivirus able to cause pallidosis First report of Beet pseudo-yellows virus on Cucurbita disease. Plant Disease 87: 1398. moschata and C. pepo in Costa Rica. Plant Disease 89: Wintermantel W.M., 2004a. Pumpkin (Cucurbita maxima and 1130. C. pepo), a new host of Beet pseudo yellows virus in Califor- Hartono S., Natkuaki T., Genda Y., Okuda S., 2003. Nu- nia. Plant Disease 88: 82. cleotide sequence and genome organization of Cucumber Wintermantel W.M., 2004b. Emergence of greenhouse white- yellows virus, a member of the genus Crinivirus. Journal of fly (Trialeurodes vaporariorum) transmitted criniviruses as General Virology 84: 1007-1012. threats to vegetable and fruit production in North Ameri- Karasev A., 2000. Genetic diversity and evolution of Clos- ca. http://www.apsnet.org/online/feature/whitefly/ teroviruses. Annual Review of Phytopathology 38: 293-324. Wisler G.C., Duffus J.E., Liu H-Y., Li R.H., 1998. Ecology Kumar S., Tamura K., Nei M., 2004. MEGA3: Integrated and epidemiology of whitefly-transmitted closteroviruses. software for Molecular Evolutionary Genetics Analysis and Plant Disease 82: 270-280. sequence alignment. Briefings in Bioinformatics 5: 150-163.

Received November 23, 2007 Accepted January 21, 2008 020_JPP_130SC_331_colore 21-07-2008 11:56 Pagina 336